Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes include, for example, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding immunoassays, a labeled ligand, including immunocompetent derivatives and analogs of the ligand is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate binding material (called a receptor herein). Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) labeled ligand. The reaction proceeds as follows:
ligand+labeled ligand+receptor.fwdarw.ligand-receptor+labeled ligand-receptor. PA1 A. contacting a liquid sample, containing the drug or a derivative thereof, with a labeled analogue of the drug or drug derivative in the presence of antibodies for the drug under conditions that promote the formation of antibody-drug immunocomplexes; and PA1 B. determining the quantity of the drug in the liquid by measuring bound or unbound labeled drug analogue; characterized in that the labeled drug analogue comprises: PA1 R.sup.2, R.sup.4, R.sup.5, and R.sup.6, each independently represents C.sub.1 to C.sub.10 alkylene or such alkylene groups interrupted with at least one or more ester groups, amide groups, --O--, --S--, and --NR--; PA1 R.sup.3 represents C.sub.1 to C.sub.3 alkylene; PA1 Z represents --O--, --S--, and --NR--, wherein R represents hydrogen or C.sub.1 to C.sub.6 alkyl, e.g. methyl propyl and hexyl; PA1 m is 0, 1, or 2; PA1 n is 0, 1, or 2; PA1 m+n&gt;0; and PA1 LABEL is an enzyme; PA1 and further provided that (i) at least one of the R.sup.1 groups is substituted or unsubstituted phenyl; (ii) one of R.sup.4, R.sup.5, and R.sup.6 can be phenylene; (iii) the bracketed components of structure I can appear therein in any order and (iv) the linking group is other than a derivative of a saturated or unsaturated monocarboxylic acid having from to 2 to 12 carbon atoms. PA1 (a) an active ester group, such as a succinimidoxycarbonyl; PA1 (b) a nucleus selected from hydantoin or barbiturate derivatives; and PA1 (c) a linking chain linking the active ester group to the hydantoin or barbiturate nucleus; wherein the linking chain is as previously defined. PA1 R.sup.7 is an ethylene or o-phenylene group. These new drug hapten analogues react with HRP, and other enzymes such as GOD and ALP, faster and more completely, than prior art analogues (b) form covalent bonds with such enzymes without adverse effect on enzyme activity, and (c) the resulting LDH analogues are more readily recognized and tightly bound by antibodies to such drugs. PA1 1) contacting a label having a nucleophilic group thereon such as an amine or sulfhydryl group, with an excess of a barbiturate or hydantoin drug hapten analogue described supra. Preferably the analogues and the label are dissolved in a water miscible organic solvent such as N,N-dimethylformamide, dimethyl sulfoxide (DMSO) or mixture of solvent and water (buffered) before mixing together; and PA1 2) removing the unused active ester and condensation by-products, preferably by dialysis. PA1 a) 1 L DMF 4'-HA/0.1M EPPS. pH 8.0, (1:1) at 42.degree. C. for 1 hr; PA1 b) Dialysis condition a) was repeated 1 time; PA1 c) 2 L 0.1M EPPS, pH 8.0, containing 0.1% BSA at 8.degree. C. for 15 hrs PA1 d) 2 L of 0.1M EPPS, pH 8.0, at 8.degree. C. for 3 hours; PA1 e) 3 L of 0.04M tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl)/0.15M NaCl, PH 7.5, at 8.degree. C. for 3 hours; and PA1 f) Dialysis condition e) was repeated 1 time for 3 hrs; PA1 a) 1 L DMF 4'-HA/0.1M EPPS, pH 8.0 (1:1) at 42.degree. C. for 1 hr; PA1 b) Dialysis condition a) was repeated 1 time; PA1 c) 1.5 L 0.1M EPPS, pH 8.0, containing 0.1% bovine serum albumin (BSA) at 8.degree. C. for 1.5 hr; PA1 d) 1.5 L 0.1M EPPS, pH 8.0, at 8.degree. C. for 18 hrs; PA1 e) 1.5 L 0.04M Tris HCl/0.15M NaCl, pH 7.5, at 8.degree. C. for 2 hrs; and PA1 f) Dialysis condition e) was repeated 1 time for 4 hrs. PA1 a) 1 LDMF 4'-HA/0.1M EPPS, pH 8.0, (1:1) at 42.degree. C. for 1 hour; PA1 b) dialysis condition a) was repeated 1 time; PA1 c) 1.5 L 0.1M EPPS, pH 8.0, containing 0.1% BSA at 5.degree. C. for 15 hours; PA1 d) 1.5 L 0.1M EPPS, pH 8.0, for 8 hours; PA1 e) 2 L 0.02M 3-morpholinopropanesulfonic acid (MOPS), pH 7.0. at 5.degree. C. for 13 hours; and PA1 f) Dialysis condition e) was repeated 2 times. PA1 a) 1 L DMF 4'-HA/0.1M EPPS, pH 8.0 (1:1) at 42.degree. C. for 1 hr; PA1 b) Dialysis condition a) was repeated once; PA1 c) 1.5 L 0.1M EPPS, pH 8.0, containing 0.1% bovine serum albumin (BSA) at 5.degree. C. overnight; PA1 d) 1.5 L 0.1M EPPS, PH 8.0, at 5.degree. C., 8 hrs; PA1 e) 2.0 L 0.02M MOPS, pH 7.0 at 5.degree. C., for at least 8 hrs; and PA1 f) Dialysis condition e) was repeated twice. PA1 (a) Polymer bead samples, each sample having one of the above-identified types of antibodies covalently bound thereto were prepared using methods and materials as described in U.S. Ser No. 081,206 filed Aug. 3, 1987 (published EPA 88 307172.2). PA1 (b) The ability of the immobilized antibodies to bind AHRP-HD 1 (label A) was determined as follows:
Conventional labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
Consistent with the foregoing an immunoassay for drugs, such as phenobarbital and phenytoin, in serum can be based on competition of an enzyme labeled analogue of such drug haptens with the drug in the patient serum for immobilized antibody binding sites.
Specific requirements for the labeled drug hapten analogues (hereafter sometimes LDH) include: 1) at least 65% of the LDH can be bound by excess immobilized antibody; 2) affinity of the LDH for immobilized antibody is such that competition of a fixed amount of LDH with the drug occurs in a therapeutically relevant drug concentration range; and 3) stability of the LDH against hydrolysis of its enzyme label under storage conditions.
Requirements imposed on the drug hapten analogue include: 1) accessibility of the analogue to the immobilized antibody following conjugation with the enzyme label; 2) specific recognition of the LDH analogue by the antibody to the drug; and 3) sufficient reactivity of the analogue with the enzyme label, either directly or following activation of the enzyme or the analogue, under conditions that do not adversely affect enzyme activity.
Glucose oxidase (GOD) and alkaline phosphatase (ALP) enzyme labels coupled to phenobarbital and phenytoin hapten analogues disclosed in U.S. Pat. 4,752,568 gave adequate enzyme labeled analogues for conducting effective competitive immunoassays in the desired format.
The problem is that the labeled phenobarbital and phenytoin analogues disclosed in the above patent were unsatisfactory for conducting competitive immunoassays when the enzyme horseradish peroxidase (HRP) was used as the label. The coupling reactions between such analogues and HRP were both slow and incomplete. Moreover phenobarbital and phenytoin HRP labels were bound very weakly so that much higher concentrations of label or antibody binding sites would be required to give a readable signal.